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					                  Original Article, EJVES 1394 (REVISION 18-3-2003)




    Markos Klonizakis, Justin M. C. Yeung, J. Roddy Nash,* Krishna Lingam,*

                           Gillian Manning & Richard Donnelly

               Division of Vascular Medicine, University of Nottingham, and

                  *Department of Vascular Surgery, Southern Derbyshire

                            Acute Hospitals Trust, Derby, UK

Short Title:             Venous insufficiency and vasodilator function

Correspondence:          Prof. Richard Donnelly MD, PhD, FRCP, FRACP
                         Division of Vascular Medicine,
                         University of Nottingham
                         Derbyshire Royal Infirmary
                         Derby DE1 2QY, UK
                         Tel: +44-1332-254966; Fax: +44-1332-254968


Objectives:     To assess the effects of posture, endothelial function and venous

insufficiency on cutaneous microvascular vasodilator function in the gaiter area, in

particular defining factors which may affect microangiopathy and ulcer formation.

Methods:        Endothelial-dependent and –independent vasodilator responses to

incremental-doses of acetylcholine (Ach) and sodium nitroprusside (SNP) were

evaluated in the perimalleolar region in the supine and standing positions in middle-

aged patients with isolated superficial venous insufficiency (ISVI) (n=25) and health

controls (n=28) using laser Doppler fluximetry (LDF) and iontophoresis of


Results: The venoarteriolar reflex (vasoconstriction on standing) was equally present

in both groups, and reduced the vasodilator responses to SNP in the upright position

(e.g for patients with ISVI, peak SNP response was 82+11 PU [standing] vs 123+15

PU [supine]). The presence of ISVI had no effect on endothelial vasodilator function

in the supine position, but on standing cutaneous reactivity to Ach was significantly

reduced (e.g peak Ach response 69+8 PU [ISVI] vs 109+11 PU [controls], p<0.003).

Conclusions: Upright posture impairs cutaneous endothelial-dependent vasodilation

in the gaiter area of patients with ISVI. This may be of clinical and prognostic utility

in identifying which patients with uncomplicated ISVI are at highest risk of tissue

breakdown and ulcer formation in the gaiter area.

Key Words: Endothelial function; microvascular; venous insufficiency; ulceration;


Gravitational ulceration around the gaiter area of the lower limb (above the medial or

lateral malleolus) due to primary venous insufficiency is a common disabling

condition that is difficult to treat once established and prone to recurrence [1]. The

underlying mechanisms of tissue breakdown and ulcer formation are complex and not

clearly understood, but microangiopathy involving structural and functional

abnormalities is an established feature of venous hypertension [2] and a possible

target for therapeutic intervention [3].

Tissue viability and susceptibility to ulceration depend upon many factors, but

cutaneous microvascular perfusion and in particular the integrity of compensatory

vasodilator mechanisms to maintain blood flow, tissue oxygenation and nutrient

delivery despite changes in systemic and local haemodynamics may be especially

important [4]. For example, it has been shown that reduced vasodilator capacity in

response to warming correlates with low transcutaneous oxygen tension in the

perimalleolar tissues [5], but very little is known about how microvascular

vasorelaxation is dependent upon endothelial function, posture (i.e neural reflex

pathways) and/or defects in the underlying vascular smooth muscle.

Although laser techniques have been available for many years, recent technological

improvements in laser Doppler fluximetry (LDF) and the associated analytical

software offer more reliable methods and applications for studying microvascular

vasodilator pathways in clinical studies, e.g by combining LDF with iontophoretic

administration of vasoactive drugs [6]. Thus, the purpose of the present study was to

compare cutaneous microvascular perfusion around the gaiter area in the supine and

upright positions in patients with isolated superficial venous insufficiency (ISVI) and

normal healthy subjects, and to assess the effects of ISVI and posture on both

endothelial-dependent and endothelial-independent vasodilator mechanisms.


Patients with uncomplicated ISVI, i.e long or short saphenous vein reflux confirmed

by Duplex scanning, were recruited from referrals to the Vascular Laboratory at

Derbyshire Royal Infirmary. Patients with present or past venous ulceration, arterial

disease, or major skin changes in the gaiter area were excluded. Healthy control

subjects were recruited from the research database in the Division of Vascular

Medicine, and the absence of ISVI among control subjects was confirmed by Duplex

scanning. All volunteers provided written informed consent, and a detailed clinical

protocol was approved by the Southern Derbyshire Research Ethics committee.

Following a baseline screening visit, which included measurements of height, weight

and BP, each subject attended the Clinical Research Unit for a single study morning

undertaken in a temperature-controlled room according to a standard protocol.

Study periods

Patients acclimatised to the room during a 30min period of supine rest with the leg to

be studied supported at a 30o angle. The gaiter area was cleaned with alcohol and

dried thoroughly before applying two perspex iontophoresis chambers (Moor

Instruments Ltd, Axminster, UK) to the surface of the leg 4-8 cm proximal to the

medial malleolus. The chambers were positioned over healthy looking skin, approx.

2-5 cm apart. 0.25ml of 1% acetylcholine (Ach) (Sigma Chemicals, UK) and 1%

sodium nitroprusside (SNP) (Nipride, Roche Pharmaceuticals Ltd) diluted in

deionised HPLC-grade water was then injected into the anodal and cathodal

iontophoresis chambers, respectively, and a laser Doppler probe positioned through

the centre of each chamber.      The chambers were no more than 4cm apart and

positioned over healthy skin (avoiding any area of lipodermatosclerosis or superficial

veins).   After achieving a stable recording of baseline flux, LDF responses to

transcutaneous administration of the endothelial-dependent vasodilator Ach and the

endothelial-independent vasodilator SNP were measured using an incremental-dose

iontophoresis protocol [7]. Thereafter, following 10 mins recovery and stabilisation

of baseline flux recordings in the standing position, repeat measurements of

vasodilator responses were performed with the subject upright.

The DRT4 LDF system was utilised for all studies (Moor Instruments, Axminster,

UK), together with the Moor Instruments iontophoresis controller and the DRT4

software for automated data analysis of skin temperature, flux and microvascular

dose-response curves for each iontophoretic challenge.

Incremental microvascular dose-response analysis

The principle of drug iontophoresis is that an electrical potential difference will

actively cause ions in solution to migrate according to their electrical charge. The

magnitude of the electrical charge (Q) is therefore dependent upon length of time (t) a

current (I) is passed (i.e Q = It, where charge is quantified in coulombs, current in

amps and time in seconds). Thus, duration and current influence the dose of agonist

that is delivered.

In this study, the iontophoresis controller (Moor Instruments) delivered low electrical

currents of preset intensity for a specified duration.     Each current stimulus was

followed by a period of laser Doppler recording to monitor the time-dependent

microvascular flow response. Following a stable baseline recording, dose-response

curves for Ach and SNP induced vasodilation were characterised using incremental

charges according to a standard protocol, as described previously [7]: 25A for 10s

(ie 250C), 50A for 10s (500C), 100A for 10s (1000C), and 100A for 20s

(2000C), with a 4-min recording period between each electrical charge.

Statistical analysis

A LDF commercial software system (Moorsoft V1, Moor Instruments, UK) was used

to provide automated analysis of the microvascular Doppler flow responses to

incremental doses of each agonist, and the data (maximum perfusion measured in

Perfusion Units [PU]) for individual subjects was then downloaded to a PC for

comparison between groups using the non-parametric Mann-Whitney U-test and

within groups using ANOVA for repeated measures. Both Clinical data and the

vasodilator responses for each incremental charge are presented as mean + SEM.


Twenty-five patients with ISVI and 28 control subjects without ISVI participated in

the study. The clinical and demographic details are presented in Table 1.

Effects of posture on endothelial-dependent & -independent vasodilator responses

In control subjects, rising from the supine to the standing position was associated with

a significant reduction in baseline cutaneous flux at both perimalleolar probes (Figure

1), typical of the normal venoarteriolar response to upright posture [8]. However, this

reduction in cutaneous perfusion appeared to be fully overcome by the endothelial-

dependent vasodilator Ach: peak microvascular responses to Ach were similar in the

standing compared with the supine position (Figure 1).         In contrast, perfusion

responses to each dose of SNP were attenuated in the standing position (Figure 1).

A similar venoarteriolar reflex was also demonstrated in patients with ISVI: baseline

cutaneous perfusion was significantly reduced when patients moved from the supine

to the standing position, and the upright response to SNP was blunted (Figure 2).

However, in contrast to the healthy controls, stimulation with Ach did not overcome

the effect of posture on cutaneous perfusion: Ach-induced vasodilation was

significantly reduced in the standing position (Figure 2).

Effects of ISVI on endothelial-dependent and –independent vasodilator responses

Baseline (resting) cutaneous perfusion in the gaiter area, both in the supine and

standing positions, was no different in patients with ISVI compared with controls.

With subjects supine (i.e without the venoarteriolar reflex active) endothelial-

dependent vasodilation to incremental doses of Ach was similar in patients vs controls

(e.g peak Ach response was 105+9 PU [ISVI] vs 121+11 PU, p=0.27) (Figure 3),

whereas the peak vasodilator response to SNP was significantly lower in the presence

of venous disease (123+15 PU vs 152+12 PU, p<0.05) (Figure 3).

When subjects moved to the standing position and the venoarteriolar (vasoconstrictor)

reflex was activated, there was no difference in vasodilator responses to SNP between

the two groups, i.e endothelial-independent vasodilation was equally blunted in

patients and controls. However, the endothelial-dependent vasodilator response to

Ach in the upright position was significantly lower in patients with ISVI (69+8 PU vs

109+11 PU, p<0.05) (Figure 3).


This study provides several important observations about the inter-play of endothelial

function, posture and vascular smooth muscle tone in the regulation of microvascular

function in control subjects and in patients at risk of perimalleolar ulceration due to

lower limb venous insufficiency. Firstly, in the supine position resting cutaneous

perfusion in the gaiter area was similar in patients with ISVI compared with controls,

and the two groups showed similar incremental responses to the endothelial-

dependent vasodilator Ach. Absolute skin flux, even in healthy individuals, is lower

in the gaiter area compared with the shin or the dorsum of the foot [9], which no

doubt explains in part why this region is so vulnerable to tissue breakdown. Although

resting perfusion is often increased over areas affected by lipodermatosclerosis or

ulceration [10], the result of this study contrasts with earlier suggestions that even

mild venous insufficiency is associated with cutaneous hyperaemia [11].

Whereas Ach-induced vasodilation in larger conduit vessels is mediated almost

entirely by endothelial release of nitric oxide (NO), the mechanisms involved in

cutaneous microvessels are less clear. It seems likely, however, that Ach-induced

release of vasodilator prostanoids and/or endothelium-derived hyperpolarising factor

(EDHF) is more important than NO [12]. Thus, under conditions of supine rest and

standardised temperature-control there was no evidence of impaired endothelial-

dependent vasodilation in patients with ISVI. A similar result was reported after

iontophoretic administration of a single-dose of pilocarpine [13]. These observations

are especially relevant to the on-going debate about what causes ISVI, in particular

suggestions that endothelial dysfunction and venous dilatation (rather than valve

failure) might be primary mechanisms in the pathogenesis of varicose veins [14].

Cutaneous vasoconstriction in response to standing is often referred to as the

venoarteriolar reflex (or ‘oedema protection reflex’) [8].      Local neuro-vascular

mechanisms, including the sympathetic nervous system, cause vasoconstriction in

response to venous hypertension and thus buffer the rise in capillary pressure and

transudation of fluid into the surrounding tissues. There have been various reports

that the venoarteriolar reflex is either intact [15] or impaired [16] in patients with

ISVI, the latter possibly due in part to a form of neuropathy affecting small

unmyelinated C-fibres in patients with venous disease [17]. In the present study,

however, ISVI had no effect on the normal vasoconstrictor response to standing.

In control subjects and in those with venous insufficiency, the venoarteriolar reflex

effectively blunted the vasodilator response at all doses of SNP, presumably by

increasing background sympathetic vasoconstrictor tone on vascular smooth muscle.

This would be consistent with noradrenergic mechanisms, activated by standing,

opposing the direct vasorelaxant effect of SNP on vascular smooth muscle. However,

it notable that upright responses to SNP were equally blunted, relative to supine

responses, in both patients with ISVI and controls, i.e this effect of posture was not

augmented or diminished by coexistent venous disease.

There were interesting differences between the groups with respect to Ach-induced

vasodilation in the upright position. Firstly, in healthy subjects, Ach stimulation

completely reversed the effect of the venoarteriolar reflex: peak vasodilator responses

to Ach in the standing position (with the venoarteriolar reflex activated) were almost

identical to those in the supine position (when the reflex was not active). This was

not the case in patients with ISVI, where maximal Ach-induced vasodilation was

significantly lower in the upright compared with the supine position.          Several

observations can be made about this result.         Firstly, from the baseline flux

measurements and the SNP data it seems unlikely that the venoarteriolar reflex was

increased in patients with venous insufficiency.     Instead, Ach-induced release of

EDHF from the microvascular endothelium, and/or EDHF responsiveness of the

vessel wall, may be reduced under conditions of raised venous pressure.

Thus, the present study has shown that there is impaired endothelium-dependent

vasodilation around the gaiter area in patients with ISVI when venous pressure is

high, e.g in the upright position. This suggests that standing is associated with

impairment of a compensatory mechanism which is probably important for

maintaining tissue perfusion and viability in venous insufficiency. Further studies

should address whether impaired Ach-induced vasodilation in the upright position is

of prognostic utility, perhaps providing a non-invasive measure of high ulcer risk

among patients with uncomplicated ISVI.


1. London NJM, Donnelly R. Ulcerated lower limb. Br. Med. J. 2000; 320: 1589-


2. Gschwandtner ME, Ehringer H. Microcirculation in chronic venous insufficiency.

Vasc. Med. 2001; 6: 169-179.

3. De Sanctis MT, Belcaro G, Cesarone MR, Ippolito E, Nicolaides AN, Incandela L,

Geroulakos G. Treatment of venous ulcers with pentoxifylline: a 12-month, double-

blind, placebo controlled trial. Microcirculation and healing. Angiology 2002; 53

(Suppl. 1): S49-S51.

4. Cheatle TR, Shami SK, Stibe E, Coleridge Smith PD, Scurr JH. Vasomotion in

venous disease. J. R. Soc. Med. 1991; 84: 261-263.

5. Cheatle TR, Stibe EC, Shami SK, Scurr JH, Coleridge Smith PD. Vasodilatory

capacity of the skin in venous disease and its relationship to transcutaneous oxygen

tension. Br. J. Surg. 1991; 78: 607-610.

6.   Morris SJ, Shore AC.      Skin blood flow responses to the iontophoresis of

acetylcholine and sodium nitroprusside in man: possible mechanisms. J. Physiol.

1996; 496: 531-542.

7. Davis KR, Ponnampalam J, Hayman R, Baker PN, Arulkumaran S, Donnelly R.

Microvascular vasodilator response to acetylcholine is increased in women with pre-

eclampsia. Br. J. Obstet. Gynaecol. 2001; 108: 610-614.

8. Henriksen O, Sejrsen P. Local reflex in microcirculation in human cutaneous

tissue. Acta. Physiol. Scand. 1976; 98: 227-231.

9. Bull R, Ansell G, Stanton AW, Levick JR, Mortimer PS. Normal cutaneous

microcirculation in gaiter zone (ulcer-susceptible skin) versus nearby regions in

healthy young adults. Int. J. Microcirc. Clin. Exp. 1995; 15: 65-74.

10. Coleridge Smith PD (1997). The microcirculation in venous hypertension. Vasc.

Med. 1997; 2: 203-213.

11. Pryce DW, Friedmann PS. Haemodynamics of leg ulceration assessed by laser

Doppler flowmetry. J. Am. Acad. Dermatol. 1993; 29: 708-714.

12.   Noon JP, Walker BR, Hand MF, Webb DJ.               Studies with iontophoretic

administration of drugs to human dermal vessels in vivo: cholinergic vasodilation is

mediated by dilator prostanoids rather than nitric oxide. Br. J. Clin. Pharmacol.

1998; 45: 545-550.

13.   Shami SK, Cheatle TR, Chittenden SJ, Scurr JH, Coleridge Smith PD.

Hyperaemic response in the skin microcirculation of patients with chronic venous

insufficiency. Br. J. Surg. 1993; 80: 433-435.

14. Clarke GH, Vasdekis SN, Hobbs JT, Nicolaides AN. Venous wall function in the

pathogenesis of varicose veins. Surgery 1992; 111: 402-408.

15. Malanin K, Vilkko P, Kolari PJ. Blood flux and venoarteriolar response of the

skin in legs with chronic venous insufficiency measured at two different depths by

using a double-wavelength laser Doppler technique. Angiology 1998; 49: 441-446.

16.   Allen AJ, Wright DI, McCollum CN, Tooke JE.                Impaired postural

vasoconstriction: a contributory cause for oedema in patients with chronic venous

insufficiency. Phlebology 1988; 3: 163-168.

17. Ardron ME, Helme RD, McKernan S. Microvascular skin responses in elderly

people with varicose leg ulcers. Age Ageing 1991; 20: 124-128.


Figure 1:   Cutaneous microvascular vasodilator responses to incremental doses of

SNP (upper panel) and Ach (lower panel) in healthy control subjects (n=28) in the

supine [     ] and standing [    ] positions; * p<0.005.

Figure 2:     Cutaneous microvascular vasodilator responses to incremental doses of

SNP (upper panel) and Ach (lower panel) in patients with ISVI (n=25) in the supine

[           ]and standing [     ]positions; * p<0.005.

Figure 3: Comparison of peak vasodilator responses to SNP (upper panel) and Ach

(lower panel) in patients with ISVI [         ]compared with controls [   ]   in the

supine and standing positions. * p< 0.05 between groups.

                               Table 1

  Clinical & demographic features of study groups. Mean & (SEM)

                               Healthy              Isolated Superficial
                               Controls             Venous Insufficiency
                                (n=28)                     (n=25)
                            Mean     Range           Mean       Range

      % Female               60%                      52%

      Age (Years)           48(11)       31 - 72     57(11)     31 – 80

      Weight (kg)           75(13)       54 - 108    79(14)     49-100

      Height (cm)          168(0.08)    157 – 188   170(0.08)   160-183

Systolic Blood Pressure    125(13)      104 – 153   140(25)     103-180

Diastolic Blood Pressure    80(10)       62 – 97     82(10)      67-97


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